Semi-continuous culture methods

Abstract

Provided herein are methods of culturing a microorganism. The methods include providing a container comprising one or more microorganisms in a medium, which has a first carbon to nitrogen ratio; culturing the microorganisms until the culture reaches a threshold indicator; harvesting a portion of the culture while maintaining the majority of the culture in the container; and adding fresh medium comprising a second carbon to nitrogen ratio to the container with the majority of the culture comprising the microorganisms.

What is claimed is: 
     
       1. A method of culturing oil-producing  Thraustochytrid  microorganisms comprising the steps of
 (a) providing a container comprising one or more oil-producing  Thraustochytrid  microorganisms in a medium comprising a first carbon to nitrogen molar ratio from 30:1 to 60:1; 
 (b) culturing the oil-producing  Thraustochytrid  microorganisms until the culture reaches a threshold indicator selected from the group consisting of cell concentration, oil content, concentration of nutrient in the culture medium, biomass productivity, oil productivity or combinations thereof; 
 (c) harvesting a portion of the culture while maintaining the majority of the culture in the container, wherein the harvested portion comprises from 5% to 20% of the culture; and 
 (d) adding to the container comprising the majority of the culture comprising the oil-producing  Thraustochytrid  microorganisms a volume of fresh medium of substantially the same volume as the harvested portion, wherein the fresh medium comprises carbon and nitrogen and a second carbon to nitrogen molar ratio from 70:1 to 95:1. 
 
     
     
       2. The method of  claim 1 , wherein the first carbon to nitrogen molar ratio is from 40:1 to 50:1. 
     
     
       3. The method of  claim 1 , wherein the second carbon to nitrogen molar ratio is from 80:1 to 90:1. 
     
     
       4. The method of  claim 1 , wherein the portion harvested comprises 10% of the culture. 
     
     
       5. The method of  claim 1 , wherein the method further comprises monitoring optical density (OD), carbon dioxide production rate, pH, dissolved oxygen (DO), time, or any combination thereof. 
     
     
       6. The method of  claim 1 , wherein the nutrient is carbon or nitrogen. 
     
     
       7. The method of  claim 1 , comprising repeating the steps of (b)-(d) one or more times after the completing the initial round of steps. 
     
     
       8. The method of  claim 1 , wherein the carbon is a carbon source selected from the group consisting of fatty acids, lipids, glycerols, triglycerols, carbohydrates, polyols, and amino sugars. 
     
     
       9. The method of  claim 8 , wherein the carbon source is glucose, fructose or glycerol. 
     
     
       10. The method of  claim 1 , wherein the nitrogen is a nitrogen source selected from the group consisting of include ammonium solutions, ammonium or amine salts, peptone, tryptone, yeast extract, malt extract, fish meal, sodium glutamate, soy extract, casamino acids and distiller grains. 
     
     
       11. The method of  claim 10 , wherein the nitrogen source is ammonium sulfate. 
     
     
       12. The method of  claim 1 , further comprising isolating oil from the harvested culture. 
     
     
       13. The method of  claim 12 , wherein the oil comprises fatty acids selected from the group consisting of alpha linolenic acid, arachidonic acid, docosahexaenoic acid, docosapentaenoic acid, eicosapentaenoic acid, gamma-linolenic acid, linoleic acid, linolenic acid, and combinations thereof. 
     
     
       14. The method of  claim 12 , wherein the oil comprises triglycerides. 
     
     
       15. The method of  claim 12 , wherein the oil comprises fatty acids selected from the group consisting of palmitic acid (C16:0), myristic acid (C14:0), palmitoleic acid (C16:1(n-7)), cis-vaccenic acid (C18:1(n-7)), docosapentaenoic acid (C22:5(n-6)), docosahexaenoic acid (C22:6(n-3)), and combinations thereof. 
     
     
       16. The method of  claim 13 , wherein the concentration of docosahexaenoic acid in the isolated fatty acids is 20% or less of the total fatty acid concentration. 
     
     
       17. The method of  claim 1 , wherein the culture is maintained at a high level of biomass productivity. 
     
     
       18. The method of  claim 17 , wherein the biomass productivity comprises 60 g/L-d to 180 g/L-d. 
     
     
       19. The method of  claim 17 , wherein the biomass productivity comprises 80 g/L-d to 130 g/L-d. 
     
     
       20. The method of  claim 17 , wherein the biomass productivity comprises 90 g/L-d to 100 g/L-d. 
     
     
       21. The method of  claim 1 , wherein the culture is maintained at a high level of oil productivity. 
     
     
       22. The method of  claim 21 , wherein the oil productivity comprises 35 g/L-d to 130 g/L-d. 
     
     
       23. The method of  claim 21 , wherein the oil productivity comprises 60 g/L-d to 90 g/L-d. 
     
     
       24. The method of  claim 21 , wherein the oil productivity comprises 70 g/L-d to 80 g/L-d. 
     
     
       25. The method of  claim 1 , wherein the culture of oil-producing microorganisms comprises a biomass comprising 60% to 85% oil. 
     
     
       26. The method of  claim 1 , wherein the culture of oil-producing microorganisms comprises a biomass comprising 65% to 75% oil. 
     
     
       27. The method of  claim 1 , wherein the culture is maintained over a period of time. 
     
     
       28. The method of  claim 27 , wherein the culture is maintained for a period of hours, days, weeks or months. 
     
     
       29. The method of  claim 27 , wherein the culture is maintained for at least 150 to 500 hours. 
     
     
       30. The method of  claim 27 , wherein the culture is maintained for at least 250 hours. 
     
     
       31. The method of  claim 27 , wherein the culture is maintained for one, two, three, four, or five weeks. 
     
     
       32. The method of  claim 1 , wherein the oil-producing microorganisms are of the genus  Thraustochytrium.    
     
     
       33. The method of  claim 1 , wherein the oil-producing microorganisms are a  Thraustochytrium  species deposited as ATCC Accession No. PTA-6245.